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Free Content Combining polymerase chain reaction restriction enzyme analysis with phenotypic characters for mycobacteria identification in Taiwan

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Abstract:

SETTING: Many hospitals use the fully-automated BACTEC 960 Mycobacteria Growth Indicator Tube (MGIT) system and acid-fast staining to detect acid-fast bacilli (AFB) in clinical specimens; however, labour-intensive biochemical methods are used for further mycobacterial species identification.

OBJECTIVE: To develop a user-friendly algorithm for mycobacterial species identification from AFB smear-positive BACTEC tubes.

DESIGN: AFB smear-positive BACTEC tubes were collected and mycobacteria were isolated and identified by biochemical methods. The tubes were subgrouped by rpoB duplex polymerase chain reaction restriction enzyme analysis (rpoB DPRA). The results were combined with key phenotypic characters of mycobacteria isolated from the tubes to develop a species identification algorithm with 16S rDNA sequencing of the isolate being used as the gold standard method.

RESULTS: By rpoB DPRA, 441 AFB smear-positive BACTEC tubes were correctly subgrouped into 100 tubes containing Mycobacterium tuberculosis complex, 335 tubes containing non-tuberculous mycobacteria and six tubes containing both. A species identification algorithm was developed by combining the rpoB DPRA results of the tubes with growth rate, photoreactivity and two biochemical results of mycobacteria recovered from the tubes.

CONCLUSION: This user-friendly algorithm can be used for mycobacterial species identification from AFB smear-positive BACTEC tubes.

Keywords: BACTEC 960 MGIT system; Mycobacterium tuberculosis complex; non-tuberculous mycobacterium; rpoB duplex PCR-restriction enzyme analysis (rpoB DPRA)

Document Type: Regular Paper

Affiliations: 1: Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan; and Department of Respiratory Therapy and Clinical Medicine, China Medical University, Taichung, Taiwan 2: Institute of Biotechnology and Chemical Engineering, I-Shou University, Kaoshiung, Taiwan 3: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan 4: Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan 5: Department of Microbiology, Taichung Veterans General Hospital, Taichung, Taiwan 6: Departments of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan 7: Departments of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan; and Rui-Fu-Shi Medical Laboratory, Taichung, Taiwan 8: Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan

Publication date: April 1, 2009

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  • The International Journal of Tuberculosis and Lung Disease publishes articles on all aspects of lung health, including public health-related issues such as training programmes, cost-benefit analysis, legislation, epidemiology, intervention studies and health systems research. The IJTLD is dedicated to the continuing education of physicians and health personnel and the dissemination of information on tuberculosis and lung health world-wide.

    Certain IJTLD articles are selected for translation into French, Spanish, Chinese or Russian. They are available on the Union website

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